Modulation of aids virus-related events by double-stranded RNAs

ABSTRACT

The use of mismatched dsRNA, e.g., AMPLIGEN® for the manufacture of compositions for use in the selective activation of a latent natural defense system within human cells, both cells already infected with AIDS virus as well as cells at risk to infection. Specific treatments for various clinical phase of the biological continuum of AIDS virus-related events ranging from subtle, early immunological lesions to advanced disease are described. Prophylaxis or prevention of AIDS virus related events, such as by introduction of mismatched double-stranded RNA into various blood products or biological fluids to be used in man (e.g., blood transfusion) or around man (e.g., dialysis programs) are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 07/433,445, filed Nov.13, 1989, now abandoned, which is a continuation of application Ser. No.07/124,572, filed Nov. 24, 1987, now abandoned, which is acontinuation-in-part of application Ser. No. 900,614 filed Aug. 26, 1986now U.S. Pat. No. 4,795,744, which in turn, is a continuation-in-part ofapplication Ser. No. 886,363 filed July 17, 1986 now abandoned, which inturn, is a continuation-in-part of application Ser. No. 769,494 filedAug. 25, 1985, now abandoned.

AIDS (Acquired Immunodeficiency Disease Syndrome) represents a majorpublic health threat of the Twentieth Century. Because of itsbiologically variable nature, its subtle means of spread from individualto individual, and its "latent" disease period which defies even theready detection of its very presence, AIDS has rapidly evolved into anepidemiologic dilemma of unprecedented proportions. Indeed, even withthe considerable resources already deployed to increase diagnosticcapabilities, the available technologies still fall well short of thenecessary requirements to gain firm control of this problem within anysection of the population (e.g., see Proceedings of Workshop entitled"Experience with HTLV-III Antibody Testing, an Update on Screening,Laboratory and Epidemiological Correlations", sponsored by the Centerfor Drugs and Biologics, FDA, National Institutes of Health, and Centersfor Disease Control, held on July 31, 1985, at the National Institute ofHealth, Bethesda, Md.; see also, Budiansky, S. in Nature, volume 316,page 96, July 11, 1985). Two different designations for the AIDS virusexist; LAV is the designator for the AIDS virus isolated at the PasteurInstitute, Paris, France and HTLV-III is the designator for the AIDSvirus isolated at the National Institute of Health, Bethesda, Md.,U.S.A. Frequently in this text, the AIDS virus will be referred togenerically or designated HTLV III or LAV without intending todifferentiate between them. Furthermore, the term "AIDS virus" in thespecification includes any and all other viruses which may be associatedwith producing AIDS, whether yet isolated or not.

Current therapeutic considerations have taken one of two approaches:immunological (vaccine production) or direct attacks on the virus itself(antiviral therapy). While vaccine production initially appearedextremely promising, at least in theory, new knowledge of the viralcomposition has significantly undermined this promise; namely, the virusapparently readily mutates or changes its basic biochemical structuresuch that critical viral antigenic "determinants"--necessary forvaccines to be effective--readily undergo mutation or change. Forexample, HTLV-III isolates in California, Maryland and England haverecently been found to be significantly different from each other whentheir genomic content(s) has been rigorously analyzed. The implicationof such studies is that vaccination against AIDS will not havewidespread lasting benefits such as the conclusively beneficial/durableresults historically characteristic of most other vaccines, such asthose directed against polio virus, measles virus, etc.

In the second, or direct antiviral approach, other scientists havetested several compounds including HPA-23, suramin, ribavirin,interferon and fascarnet, etc. These compounds have been introduced intoeither the laboratory or clinical studies with little-to-no evidence oftherapeutic success to date. Indeed, consistent evidence of hightoxicity and/or severe side-effects has been reported in nearly everyinstance (e.g., see summary prepared by M. Clark, Newsweek, page 71,Aug. 5, 1985; also, C. Wallis, Time, pages 40-47, Aug. 12, 1985). Noneof these drugs are indeed new or able to be selectively directed againstthe underlying disorder; namely, multiplication of AIDS virus in certaincells. For example, HPA-23 is a combination of heavy metals (reminiscentof the use of arsenic to treat venereal diseases in the 1920s, orpre-penicillin era); suramin is actually an anti-parasitic (sleepingsickness) compound and fascarnet is an anti-herpes virus compound. Thelatter two compounds may inhibit an AIDS-virus related enzyme termed"reverse transcriptase", but there is no evidence that such an enzymaticinhibition, in fact, would result in any therapeutic improvement ordisease prevention/amelioration. Similarly, interferon, which is astoxic and non-specific as the above-mentioned compounds, may have somelimited activity on AIDS-virus related tumors, but numerous studiesindicate that it holds little promise as an effective antiviral againstAIDS virus, or most other viruses for that matter.

Mismatched double-stranded RNA is a known form of macromolecular RNA(see U.S. Pat. Nos. 4,024,222 and 4,130,641) in which destabilization ofthe double helix prevents base pairing. Mismatched dsRNA is well knownfor its interferon-induction properties which indicate a mechanismunrelated to interferon induction per se (e.g., see European PatentApplication 83305426.5 filed Aug. 15, 1983, entitled "AntiproliferativeAction of dsRNAs on Tumor Cells"). A typical therapeutic embodiment ofmismatched double-stranded RNA is the synthetic dsRNA formed bycomplexes of polyriboinosinic and polyribocytidylic/uridylic acid, suchas rI_(n) r(C_(x),U or G)_(n) where x has a value from 4 to 29, e.g.rIn.r(C₁₂ U)_(n) herein referred to as "Ampligen", a trademark of HEMResearch, Inc., of Rockville, Md., U.S.A. Many mismatched dsRNA polymerswhich behave similarly to Ampligen have been studied. The keytherapeutic advantage of mismatched dsRNAs over other forms of naturaland/or synthetic dsRNAs is their reduced toxicity in animals and man.For example, Lampson et al in U.S. Pat. No. 3,666,646 described earliercomplexes of dsRNA which are capable of triggering variousinterferon-related effects, but the toxicity of such compounds precludedany clinical utility in the treatment of cancer or related disorders.

The inventor has fully described the known activities of variousinterferons, interferon inducers and dsRNAs in his recent textbooks onthe subject (e.g., see the inventor's chapter in Anticancer andInterferon Agents, pages 301 through 319, edited by R. M. Ottenbrite andG. B. Butler, Marcell Dekker, New York, 1984; also see Handbook ofExperimental Pharmacology on Interferons, edited by P. E. Come and W. A.Carter, 1984, published by Springer-Verlag, New York and Heidelberg;pages 535-555 fully describe the known properties of dsRNAs).

Mismatched dsRNA, e.g., Ampligen, is now known to have therapeuticactivity against certain human tumors, particularly kidney cancer.Although currently thought of as purely an "interferon inducer", dsRNAsare active on certain human tumors which are completely resistant tointerferon itself as fully described in European Patent Application83305426.5 of which the present applicant is an inventor.

Similar to the lack of activity against certain tumors, it has now beendemonstrated that virtually all types of interferon exhibit nosignificant activity in the treatment of the AIDS virus. Being known"interferon inducers", it is particularly surprising that mismatcheddsRNAs have application in the treatment of AIDS-related disorders.

The present invention is based on another new and unexpected property ofdsRNA, especially mismatched dsRNA, as exemplified by Ampligen, whichhas a surprisingly wide range of applicability to the treatment ofAIDS-related disorders.

The present invention provides the use of a dsRNA in the manufacture ofa composition for use in the treatment of AIDS by therapy orprophylaxis.

This invention includes a method of treating retrovirus or T-celllymphotropic virus-induced conditions, including AIDS, in a personinfected with same, by administering to that person an amount of dsRNAsufficient to therapeutically benefit AIDS-related disorders. A secondvirus inhibitor, such as interferon, may be concurrently administered tothe patient.

An important symptom of AIDS is the suppression of the normal immunestate as indicated by the immune skin response. This is restored duringsuccessful treatment with dsRNA.

Accordingly, the invention includes the use of dsRNA in the manufactureof a medicament for restoring an anergic immune state to a substantiallynormal immune state as measured by testing the immune skin response.

The dsRNA may be a mismatched dsRNA.

HTLV III/LAV infection produces a particularly wide range of severity ofresponse from relatively asymptomatic carriers to profound immunologicalparalysis. To treat this range of response, a wide range of dosage ofdsRNA may be appropriate. When low dosages are employed, the use ofnon-mismatched dsRNA may be appropriate and dosages may be sufficientlylow to avoid the adverse reactions mentioned above. Where higher dosesare appropriate the need to use mismatched dsRNA will be greater.

Preferably, the dsRNA is such as to produce an elevated intracellularconcentration of 2'-5'A oligomers without host toxicity, e.g. due to anaccelerated bioavailability, a structure adapted to activate 2'-5'Aoligomer production without producing side effects, or due to arelatively short half life following administration.

By "matched dsRNA" are meant those in which hydrogen bonding (basestacking) between the counterpart strands is relatively intact, i.e. isinterrupted on average less than one base pair in every 29 consecutivebase residues. The term "mis-matched dsRNA" should be understoodaccordingly.

The dsRNA may be a complex of a polyinosinate and a polycytidylatecontaining a proportion of uracil bases or guanidine bases, e.g. from 1in 5 to 1 in 30 such bases (poly I. poly (C4-29×U or G).

The dsRNA may be of the general formula rI_(n).C(C₁₂ U)_(n). Othersuitable examples of dsRNA are discussed below.

The composition may comprise the dsRNA and an AIDS inhibition assistingagent, such as a lymphokine, e.g. an interferon or an interleukin suchas IL-2.

The invention accordingly includes the use of dsRNA in the manufactureof a pharmaceutical composition for the prevention or treatment of AIDSin a person comprising, in combination, a dsRNA and a lymphokine such asan interferon.

Preferably the composition contains the dsRNA and the interferon in theratio of 0.01 to 1000 micrograms of dsRNA to 0.1 to 100,000 IRU ofinterferon.

The invention further includes a method of rendering a, preferablyhuman-originating, biological fluid or, preferably human-originating,cells resistant to viral infection, enhancing the resistance thereof, ormitigating the effects of infection therefrom comprising admixing orcontacting said biological fluid or cells with an AIDS-inhibiting amountof a dsRNA.

The biological fluid may be human blood or a fraction thereof forinstance for use in transfusion or dialysis.

The invention further includes as a composition of matter, human bloodor a fraction thereof or human cells and an AIDS-inhibiting amount of amismatched dsRNA.

Furthermore, the invention includes also a process of removing orinactivating AIDS viruses from a device for handling parenteral fluidsor mitigating the effect of infection therefrom comprising contactingsaid device with a composition containing an AIDS-inhibiting amount of adsRNA.

The invention includes also a composition for use in the treatment ofAIDS comprising a dsRNA and a pharmaceutically acceptable carrier ordiluent together with instructions for use in the treatment of AIDS.

Thus there are disclosed hereinafter by way of example, therapeuticmethods of selectively inhibiting human viruses, specifically the AIDSvirus, without significant toxicity to normal cells; methods ofinhibiting, delaying or preventing a person from becoming infected withAIDS virus; methods of preventing or treating AIDS-related disorders inhumans; methods of treating human biological fluids, cells and tissueproducts to prevent or arrest infection or contamination with AIDSvirus; and methods of correcting specific lesions associated with AIDSvirus, including loss of interferon receptors on cells within the immunesystem, cutailment or reduction of intracellular 2'-5'A synthetase andthe reduction or loss of intracellular dsRNAs in selected cells of theimmune system. Pharmaceutical compositions for use in these methods arealso disclosed.

The use of dsRNA is adapted to correct specific lesions associated withAIDS virus, including loss of interferon receptors on various criticalcells within the immune system, curtailment/reduction of intracellular2'-5'A synthetase in various critical cells within the immune/bodilydefense system, and reduction or loss of intracellular dsRNA in variouscritical cells within the immune/bodily defense system.

The mismatched dsRNAs preferred for use in the present invention arebased on copolynucleotides selected from poly (C_(n),U) and poly(C_(n),G) in which n is an integer having a value of from 4 to 29 andare mismatched analogs of complexes of polyriboinosinic andpolyribocytidilic acids, formed by modifying rI_(n). rC_(n) toincorporate unpaired bases (uracyl or guanine) along thepolyribocytidylate (rC_(n)) strand. Alternatively, the dsRNA may bederived from poly (I). poly (C) dsRNA by modifying the ribosyl backboneof polyriboinosinic acid (rI_(n)) e.g. by including 2'-O-methyl ribosylresidues. These mismatched analogs of rI_(n).rC_(n), preferred ones ofwhich are of the general formula rI_(n). r(C₁₂,U)_(n), are described byCarter and Ts'o in U.S. Pat. Nos. 4,130,641 and 4,024,222 thedisclosures of which are hereby incorporated by reference. The dsRNA'sdescribed therein generally are suitable for use according to thepresent invention.

Other examples of mismatched dsRNA for use in the invention include:

poly (I). poly (C₄, U)

poly (I). poly (C₇, U)

poly (I). poly (C₁₃, U)

poly (I). poly (C₂₂, U)

poly (I). poly (C₂₀, G)

poly (I). poly (C₂₉, G) and

poly (I). poly (C_(p)) 23 G>p

Pharmaceutical compositions in accordance with this invention includethe mismatched dsRNA, optionally also an interferon, together with apharmaceutically acceptable carrier or diluent. Pharmaceuticalcompositions contemplated by this invention include those adapted forparenteral administration in a suitable pharmaceutical vehicle.

Thus, for example, parenteral solutions, suspensions and dispersions canbe prepared as required according to known pharmaceutical techniqueswith sterile or pyrogen-free water as the solvent/diluent optionallyalso with physiologically acceptable salts.

It will be understood that the absolute quantity of active ingredientspresent in any dosage unit should not exceed that appropriate to therate and manner of administration to be employed, but on the other handshould also desirably be adequate to allow the desired rate ofadministration to be achieved by a small number of doses. The rate ofadministration will moreover depend on the precise pharmacologicalaction desired.

The amount of mismatched dsRNA administered is preferably sufficient toachieve a level of from 0.01 micrograms per millilitre of body fluidafter equilibration of the dsRNA level through the body fluid up to 1000micrograms per milliliter in the systemic circulation immediatelyfollowing administration. When concurrently administered, interferon ispreferably included in an amount that results in a level of 0.1 to100,000 IRU per milliliter of body fluid. Generally, the amount to beadministered will depend on the severity of the condition, in particularthe level of available intracellular bioactive dsRNA, 2'-5' oligo Amolecules or 2'-5'A synthetase. As used herein, the term body fluid isthe solution of serum, vitamins, etc. which circulates within theorganism and bathes the tissues. Expected body fluid volumes of patientsare of course known to practitioners and are published as charts andtables, which are routinely available.

As indicated above, the invention includes the treatment of biologicalfluids with dsRNA and similarly the treatment of cells with dsRNA. Bloodproducts such as whole blood used in transfusions or components of wholeblood, such as "packed (concentrated) red blood cells, packed whiteblood cells, platelet concentrates or serum protein fractions such asimmunoglobulins" may be treated in this way. Such blood products mayhave mismatched dsRNA at appropriate concentrations added to them at thetime of initial isolation and prior to any cryopreservation.

Alternatively, an effective concentration may be added to the bloodproduct immediately prior to injection into the recipient. In suchcases, the operator may simply refer to a standard table of body fluidvolumes which interrelate the weight of the recipient to his or her bodyfluid volume, which is the total of the body fluid volume of the patientand the body fluid volume available for equilibration with the necessaryquantity of the dsRNA. A sixty to seventy kilogram patient will have abody fluid volume of approximately five to six liters.

Blood products as described above may have undetected retroviruses,especially AIDS-related virus associated with them. The objective is toprovide a final concentration in the blood product in question whichprevents the "seeding" of the occult retroviruses in the diverse tissuesof the recipient of the blood product. By preventing a successfulseeding of such retrovirus one can prevent what otherwise would become alife threatening viral proliferation when the contaminated transfusedcells and/or cell products are admixed and/or distributed throughout therecipient's system.

Other important uses for this technique are instances where thepatient's blood is transitorily exposed to donor blood and/or bloodproducts, such as through a membrane which may be defective or may serveto transmit retroviruses, examples of such instances include the use ofequipment such as extracorporeal pumps and associated devices usedduring cardiac surgery, cardiac bypass surgery, organ transplants and soforth. This differs from the situation in which whole blood or bloodproducts are infused into the patient. Transitory exposure through amembrane involves only temporary exposure to possibly contaminatedblood, exposure lasting as little as a few minutes or for as long asseveral hours depending on the length of the surgical or other medicalprocedure. Similar exposure is possible with renal dialysis equipment inwhich the presence of an effective concentration of dsRNA will thwartthe possibility that undetected retroviruses may traverse membranes andseed the body of an individual connected to such equipment.

Medical literature also documents the passage of the AIDS-related virusthrough nasal and lachrynal secretion. Medical equipment and deviceshaving the potential to transfer retroviruses from one patient toanother are also effectively treated with mismatched dsRNAs according tothe invention. Illustrative examples include inhalation and inhalationtherapy equipment used to assist in respiration as well as instrumentsused to examine and treat eyes.

Aerosols containing dsRNAs may be sprayed onto the contacted portions ofthe device to prevent contamination of equipment due to multiple users.

The amount of dsRNA included in or added to whole blood or bloodproducts will depend on the overall dilution rate of the blood or bloodproducts in use. First, the tissue volume of the patient may bedetermined based upon the size of the patient, then the quantity ofdsRNA may be calculated. A transfused blood product will require agreater concentration of the dsRNA because of the extensive overalldilution as compared with an extracorporeal blood pump or dialysisequipment using a membrance in which the donor patient is to beprotected without concern for dilution to immobilise any retrovirus inthe donated material. Preferred amounts for non-diluted uses are in therange of from about 0.1 microgram to about 200 micrograms per milliliterof body fluid. In contrast, for whole blood, usually available incontainers or 350 and 500 ml, a sixty kilogram patient with a whole bodyfluid volume of between about five and six liters will require about 200mg of dsRNA to achieve a concentration of 40 micrograms per milliliterupon equilibration.

The invention will be illustrated by the following description ofexamples of the use of dsRNA.

Mismatched dsRNAs, e.g., Ampligen, were formulated in aqueous solutionsuch that, when added to various human cells, transient concentrationsof 0.01 microgram to 250 microgram per milliliter of fluid (bathingcells) was obtained. A variety of different cells, all potential targetsfor infection with HTLV-III (AIDS virus), were used.

Below described is a typical experiment with H-9 cells, a human lymphoidcell which can be acutely or chronically infected with HTLV. The cellswere grown under standard conditions (e.g., see Mitsuya et al, Sciencevolume 226, page 172, 1984) and analyzed for several weeks with respectto the presence of HTLV enzymes or HTLV-specific proteins. Variousconcentrations of Ampligen were added, before, after or simultaneouslywith the virus to thus mimic various clinical conditions. Mostimportantly, careful analyses of cell number, morphology, etc., weredone to determine if the mismatched dsRNA had various non specificeffects on lymphoid cell growth as had been described by Mitsuya withother inhibitors. Cells, at different times during the experiment, wereisolated and studied by HPLC analysis of the 2',5'-A oligomers usingmethods described by Lee et al (Biochemistry, volume 24, page 551,1985). Certain A oligomers, when present in nature, are known to conferviral resistance to cells, but it has not been previously described, oranticipated, that a test compound (synthesized by man) could preciselytrigger this natural reaction selectively, and thus strengthen a naturaldefense mechanism against viruses in general, and AIDS virus inparticular.

                  TABLE 1                                                         ______________________________________                                        Effect of Ampligen on HTLV-III Infectivity                                            % HTLV                                                                        Positive Cells      Reverse Transcriptase                             Day     + Drug   - Drug     + Drug  - Drug                                    ______________________________________                                        Experiment A                                                                  4       0        50         1,412     24,287                                  9       7        90         144,632   1,243,300                               Experiment B                                                                  3       0        0          353       323                                     7       0        0          400       1,200                                   10      0        5          800       2,261                                   14      2        >80        1,100     112,247                                 17      5        >90        1,200     1,560,000                               ______________________________________                                    

Experiment A was conducted with 25 times more virus (expressed asinfectious units) than target cells whereas Experiment B was conductedwith 10 times more cells (designated H-9 cells) than virus. Percentcells positive refers to cells which expressed HTLV-III markersdesignated p24 and p19 as determined by immunofluorescence; reversetranscriptase refers to a viral enzyme measured in the cell supernatantby the standard template designated poly rA/dT. Concurrently, cellcounts were done: the cells multiplied at normal growth rates at allconcentrations of Ampligen tested (up to 300 micrograms per milliliter).In experiments A and B, Ampligen (50 micrograms per milliliter) wasadded on day 1. HPLC analysis showed (by day 3-7 in either Experiment Aor B) a specific shift in the 2',5'-oligomer profile such that thehigher (less antiviral) molecular weight (MW) oligomers shifted to thelower (most antiviral) MW oligomers which contributed to a selective andstrong suppression of the AIDS virus.

Similar experiments were conducted with various other human cells whichare potential in vivo targets for AIDS virus: These experiments includedother cells functionally determined to be NK (natural killer) cells, Thelper cells, T suppressor cells and mononuclear cells, etc. In allinstances, various concentrations of mismatched dsRNA were able toselectively arrest and/or prevent HTLV multiplication without any effecton normal cell growth and maturation. Proliferation of the T cells inculture was maintained by the standard addition of IL-2 factor as wellknown to those familiar in the art of cell biology. The selectivesuppression of HTLV by mismatched dsRNA was consistently associated witha specific shift, or enhancement, in the 2',5'-oligomer profile asdetermined by HPLC analysis.

COMPARISONS OF MISMATCHED dsRNAs WITH INTERFERON

Since dsRNAs, being "interferon inducers", might be viewed as simplyworking through (via) an interferon mechanism, any activity ofinterferon per se might be thought to suggest activity by dsRNA. Thus,the following experiments establish that uniqueness of the presentinvention by demonstrating no significant activity of interferons ofvirtually all types in the face of very potent and specific anti-AIDSvirus activity by Ampligen.

CEM (another human T cell line) cells were treated with either 250I.U./ml recombinant alpha interferon, 250 I.U./ml natural betainterferon, 50 I.U./ml natural gamma interferon of 50 μg/ml Ampligen for18 hours prior to infection with LAV. After 15 days of culture, thefollowing data were collected.

                  TABLE 2                                                         ______________________________________                                        Effect of Ampligen on LAV Infectivity                                                  Reverse tran-                                                                             indirect immunofluores-                                           scriptase [cpm/ml                                                                         cence [% of cells with                                            culture fluid]                                                                            LAV antibody binding]                                    ______________________________________                                        untreated  1,147,000     49%                                                  alpha interferon                                                                           503,000      5%                                                  beta interferon                                                                          1,500,000     60%                                                  gamma interferon                                                                           360,000     30%                                                  Ampligen     24,000      <1%                                                  ______________________________________                                    

The data indicate a profound inhibition of viral replication byAmpligen.

Thus, dsRNA (Ampligen) inhibits (>99%) HTLV-III/LAV infection in H9 (T4)cells and CEM (T4) cells. Interferons alpha, beta and gamma aremarginally active or inactive. Viral inhibition by Ampligen is veryselective and occurs without any measurable effect on cell growth.

Thus, Ampligen inhibits HTLV-III/LAV virus replication by a mechanismdistinct from interferon. This finding suggests that Ampligen andinterferon or other drugs could be used in combination, whereby Ampligenwould inhibit viral replication and the interferon could be used instimulating the immune system to restore pre-disease function.

In the restoration of the natural antiviral state Ampligen acts distalto the AIDS/ARC block in the antiviral pathway to restore a naturalantiviral RNase activity which destroys the viral genome.

Therefore, the drug has the potential to inhibit viral replication, aswell as to augment the immune response. Thus, it should be noted thatAmpligen is not simply a reverse transcriptase inhibitor of the virus,but acts through the dual mechanisms of human immune system stimulationand establishment of an antiviral state in target cells. Ampligen isreadily set apart from interferons, in that it may well be able to cureviral infections and viral induced tumors in animals which arecompletely refractory to exogenous interferon therapy. Theseobservations distinguish Ampligen from many other drugs proposed for useagainst this disease.

In related studies, the pathophysiology of AIDS was studied andunexpected and previously undetected biochemical lesions in individualspredisposed (homosexual) to AIDS were detected. Such lesions arecorrectable by Ampligen and explain its potent antiviral activity in theface of nominal activity (if any), by interferons or most othercompounds which have been tested.

First, it was observed that the missing critical biochemical co-factorwas dsRNA, in the T lymphocytes of each of 6 individuals predisposed toAIDS (homosexual males) and in each of 6 AIDS victims. Measurements weremade of dsRNAs by standard techniques following cell disruption. Theeffect of Ampligen can thus be accurately viewed as specific replacementtherapy for a critical biochemical entity needed for humans to withstandvirus attack at the subcellular level. When dsRNA is present naturallyor added, as via Ampligen therapy, an active RNase L is formed whichthen "eats-up" (hydrolizes), or destroys, the viral mRNA thus stoppingor containing the viral infection. Subsequently to the filing of the USApriority application, Preeble et al reached a complementary conclusion(September 1985 issue of Journal of Infectious Diseases, pages 457-465,entitled "Interferon Induced 2'-5' Oligoadenylate Synthetase DuringInterferon Alpha Therapy in Homosexual Men with Kaposi's Sarcoma: MarkedDeficiency in Biochemical Response to Interferon in Patients withAcquired Immunodeficiency Syndrome"). They showed that the addition ofinterferon to immune cells from AIDS victims did not result in theexpected boost in this specific intracellular pathway, a first line ofdefense against various viruses.

Second, it was determined that AIDS virus infection itself caused afurther loss, or alteration, in the interferon receptor on the cellsurface such that interferon could not bind well and could not thereforeinititiate the critical "cascade" of biochemical events normally leadingto arrest of viral infection and restoration/maintenance of immunecompetence. It was further observed that unusually low levels of 2'-5'Asynthetase follow HTLV-III infection. Thus, AIDS virus infection itselfis characterized by applicant at least three (3) deleterious eventswhich however, can be specifically remedied by Ampligen or dsRNAtherapy: (1) loss of interferon receptors, (2) reduction in 2'-5'Asynthetase, and (3) abnormally low intracellular dsRNA.

In the relevant biochemical pathway, dsRNA works downstream or distal tothe biochemical lesions in individuals at risk (with or without activeAIDS virus infection), thus restoring normal antiviral responsecapability.

dsRNA is very efficiently taken up by cells and therefore does notrequire an intact IFN receptor. Further, mismatched dsRNA is alsoavailable as bioactive fragments to accelerate intracellular uptake anddistribution. Thus, dsRNA has the potential to readily cross theblood-brain barrier, which may be a residual reservoir of AIDS virus insome individuals.

Typically, dsRNA for use in the present invention will be of themolecular size exemplified in U.S. Pat. Nos. 4,024,222 and 4,130,641 butmuch lower molecular weight dsRNA, obtainable as fragments of the parentmolecule are also effective. These may for instance be of one half toone tenth the size of the typical dsRNA materials described previously.

dsRNA activates the 2'-5'A polymerases and promotes the synthesis of a500-fold increment in the active antiviral oligonuleotides over thatpossible with interferons alone (dsRNA leads to 250 nanomoles of 2'-5'oligo A in 1.6×10⁸ cells whereas 200 units/ml IFN lead to only 0.5nanomoles of 2'-5' oligo A.

Similar phenomena were observed by applicant in the NK cells ofindividuals at risk to development of AIDS, or possessing frank AIDS. Asimilar situation exists with respect to the levels of Natural Killercell (NK) activity in AIDS/ARC patients. However, much less is knownregarding the mechanisms of NK regulation.

AIDS/ARC patients and healthy members of "at-risk" groups often haveweak immunosurveillance capacity (functional NK and T lymphocytes) andcannot be re-activated by interferons. Ampligen acts distal to thedisease block and can activate cytotoxic lymphocytes.

The above theories have been tested clinically in the followingconfidential trial.

A 60 kg adult male with an AIDS-related complex of intermediate severity(lymph nodes enlarged and unable to eat solid foods for almost one yeardue to enlarged lymph nodes, AIDS-virus concentration greater than 10⁵particles per ml of blood but having no evidence of tumors or otherinfections) was treated with 200 mg mismatched dsRNA (Ampligen) admixedwith physiologic saline solution by intravenous drip over a period of 30minutes. This infusion resulted in a concentration of approximately 40mcg (0.04 mg) per ml of body fluid when the dsRNA was completelycirculated throughout the body and fully equilibrated with all of theextracellular body fluid. The dsRNA was infused into the patient on 6consecutive treatment intervals spaced 2-3 days apart and was sufficientto rid the patient's body of all measurable AIDS-related virus and tocorrect the patient's deteriorated immune function. Restoration of theimmune function was indicated by a 50% improvement in the ratio of T4/T8lymphocytes, a ratio recognized as a reliable measurement of generalimmune capability with respect to retroviruses. Further, at theconclusion of therapy the patient's immune capability, as measured byskin testing, returned to normal from the anergic condition that wasmeasured prior to administration of the mismatched dsRNA. The patientwas able to resume eating solid food at the conclusion of therapy andhis conditions continues to progress.

Therapeutic combinations of mismatched dsRNAs and lymphokines exhibitenhanced effectiveness in inhibiting viral (HIV) replication.

Table 3 shows the synergistic activity of dsRNA and lymphokines on viralreplication. A retrovirus (HIV) was exposed to graded doses ofrecombinant beta interferon without or with dsRNA. Normally, the viralinfection caused extensive cytolysis but dsRNA at very low doses (12.5micrograms/ml) causes substantial residual protection even after 5 daysof viral replication and also had a pronounced and unexpected enhancingeffect on the activity of interferon. Similar experiments summarized inTable 4 demonstrate the ubiquity of the effect both with respect toother immune system components (cells) and other lymphokines.

Lymphokines of particular interest include the interferons, as discussedabove, both naturally occurring and made recombinant technology. Alsothe interleukins, specifically interleukin-2 (IL-2) and recombinantinterleukin-2 (rIL-2), and tumor necrosis factor (TNF) may beconsidered. Also included are lymphokine activated killer cells (LAK)formed in animals in response to exposure to a lymphokine.

When interferon (alpha) is used as the lymphokine, an amount of from0,01 to 100,000 IRU per milliliter of the patient's body fluid isprovided. When IL-2, preferably rIL-2, is the lymphokine, the amountadministered lies within a range of about 10² IL-2 units per kg of thepatient's body weight up to a value approaching unacceptable levels oftoxicity in that patient, which may be as high as 10⁶ IL-2 units.However, most effective, toxic-reaction manageable values are in therange of from about 10³ to about 10⁴ IL-2 per kg of body weight.

                  TABLE 3                                                         ______________________________________                                        Anti-HIV Activity of Combined rIFN-β and Ampligen                        Treatment in T-Cell Cultures                                                           Percent Protection*                                                  rIFN-β                                                                            4 Day           5 Day                                                (Units/ml)                                                                             -Ampl    +Ampl.sup.±                                                                           -Ampl  +Ampl                                     ______________________________________                                        1250     58       100        19     64                                        625      42       100        8      55                                        312      31       94         4      44                                        156      31       90         2      44                                        78        8       57         2      25                                        39        5       46         0      17                                        19.5      2       24         0      11                                        9.7       0       22         0       5                                        ______________________________________                                         *Average values, n = 3. Standard deviation were always within 10% of          values.                                                                        Cells were incubated in the presence or absence of rIFNβ (Triton        Biosciences, Inc.) and Ampligen for 4 hours prior to virus challenge.         Effectors were assayed in 96well microtiter plates using MT2 as target        cells and HTLVIIIB as virus at an M.O.I. of 0.1-1.0.                          .sup.±Ampligen = 12.5 μg/ml. Percent protection provided by Amplige     alone: 4 day, 31%; 5 day, 3%.                                            

                  TABLE 4                                                         ______________________________________                                        Inhibition of HIV Replication by rIFN-α A                               and rIFN-β ser 17 with and without Ampligen                                       cpmRT/ml   Avg. cmpRT/ml                                             Effector*                                                                              (×10.sup.-3)                                                                       (×10.sup.-3)                                                                         % Decrease                                   ______________________________________                                        Control-1                                                                              877        910          --                                           Control-2                                                                              943                                                                  Ampligen-1                                                                             896        852           6                                           Ampligen-2                                                                             807                                                                  rIFNαA-1                                                                         426        428          53                                           rIFNαA-2                                                                         430                                                                  rIFNβ-1                                                                           396        398          56                                           rIFNβ-2                                                                           400                                                                  Ampligen +                                                                              41         47          95                                           rIFNαA-1                                                                Ampligen +                                                                              54                                                                  rIFNαA-2                                                                Ampligen +                                                                              41         44          96                                           rIFNβ-1                                                                  Ampligen +                                                                              48                                                                  rIFNβ-2                                                                  ______________________________________                                         *Equal density cultures of H9/HTLVIIIB were incubated in duplicate in the     presence and absence of effectors. Conditioned culture fluids were            harvested after 48 hours and assayed for reverse transcriptase activity.      No inhibition of cell division was observed.                                  IFNs = 500 μ/ml                                                            Ampligen = 50 μg/ml                                                   

The combination of a dsRNA and a lymphokine, administered separately,including prior administration of the lymphokine to "prime" the relevantcells, or concurrently, protects cells of the immune system from viralinfection. The degree of protection conferred by the combination isgreater than that conferred by the dsRNA alone. Importantly, thecombination of agents causes no deletarious inhibition of the normalcell division process.

AMPLIGEN® is a registered trademark of HEM Research, Inc., USA.

What is claimed:
 1. A method of treating retrovirus or T-celllymphotropic virus-induced conditions including AIDS in a personinfected with same comprising administering to that person an amount ofdsRNA sufficient to therapeutically benefit AIDS-related disorders. 2.The method of claim 1 in which the dsRNA is mismatched dsRNA.
 3. Themethod of claim 2 in which the dsRNA is a complex of a polyinosinate anda polycytidylate containing of from 1 in 5 to 1 in 30 uracil orguandidine bases.
 4. The method of claim 3 in which the mismatched dsRNAis rI_(n) ·(C₁₂ U)_(n).
 5. The method of claim 1 in which the dsRNA isadministered in an amount sufficient to result in a level of 0.01 to1000 microgram per milliliter of the patient's body fluid.
 6. The methodof claim 1 in which an AIDS inhibitor is administered concurrently withthe dsRNA.
 7. The method of claim 1 in which a second AIDS virusinhibitor is concurrently administered to the patient together with thedsRNA.
 8. The method of claim 1 in which a lymphokine is administeredwith the dsRNA as an AIDS inhibition assisting agent.
 9. A method oftreating AIDS in a person infected with same comprising administering tothat person, in combination, an amount of mismatched dsRNA sufficient tobenefit AIDS-related disorders and an interferon or an interleukin in anamount sufficient to assist in inhibiting AIDS.
 10. The method of claim9 in which the mismatched dsRNA and the interferon are administered inan amount sufficient to result in a ratio of 0.01 to 1000 microgram permilliliter of dsRNA to 0.1 to 100,000 IRU of interferon in the patient'sbody fluid.
 11. A therapeutic composition for the prevention andtreatment of retrovirus or T-cell lymphotropic virus including AIDS in aperson comprising, in combination, a viral-inhibiting amount of dsRNA incombination with an AIDS-inhibiting amount of interferon, the dsRNApresent in an amount when administered to a person to result in a levelof 0.01 to 1000 micrograms per milliliter of body fluid and theinterferon present in an amount when administered to a person to resultin a level of 0.1 to 100,000 IRU per milliliter of body fluid.
 12. Thetherapeutic composition of claim 11, in which the dsRNA is mismatcheddsRNA.
 13. The therapeutic composition of claim 11 in which themismatched dsRNA is rI_(n) ·(C₁₂ U)_(n).
 14. A method of rendering ahuman-originating biological fluid or cells resistant to infection withretrovirus or T-cell lymphotripic virus including AIDS virus, enhancingthe resistance thereof, or mitigating the effect of infection therefromcomprising admixing or contacting said biological fluid or cells with avirus-inhibiting amount of dsRNA.
 15. The method of claim 14 in whichthe dsRNA is mismatched dsRNA.
 16. The method of claim 14 in which themismatched dsRNA is rI_(n) ·(C₁₂ U)_(n).
 17. The method of claim 13 inwhich the biological fluid is human blood or a fraction thereof, or thecells are NK cells, T-cells, T-suppressor cells, T-helper cells,spermatozoa, ova or fetal cells.
 18. As a composition of matter, humanblood or a fraction thereof or human cells and an effective amount of amismatched dsRNA.
 19. A process of removing or inactivating retrovirusesor T-cell lymphotropic viruses including AIDS viruses from a device forhandling parentheral fluids or mitigating the effect of infectiontherefrom comprising contracting said device with a compositioncontaining an AIDS-inhibiting amount of a dsRNA.
 20. A method ofrestoring an anergic immune state to a substantially normal immunestate, as measured by skin testing, said method comprising administeringto a person having an anergic immune state an effective amount of dsRNA.